The present invention relates to the superfamily of nuclear receptors known as the steroid/thyroid hormone receptors and their cognate response elements. More particularly, the present invention relates to the discovery of novel response elements which may be used to control the transcriptional activity of promoters.
A central question in eukaryotic molecular biology is how specific DNA-binding proteins bind regulatory sequences to influence cell function and fate. The steroid/thyroid hormone receptors form a superfamily of ligand-dependent transcription factors that are believed to play a part in such cell function and fate. For example, it is known that these receptors transduce extracellular hormonal signals to target genes that contain specific enhancer sequences (referred to as hormone-response elements, or HREs). Each receptor contains a ligand-binding domain and a DNA-binding domain. The receptor undergoes a conformational change when it binds ligand. This conformational change permits the receptor-ligand complex to bind its cognate response element and thereby regulate transcriptional activity of an associated promoter. Transcriptional activation of promoter drives transcription of an operatively associated structural gene.
Sequence comparison and mutational analyses of hormone receptors, such as the glucocorticoid receptor (GR), have identified functional domains responsible for transcriptional activation and repression, nuclear localization, DNA binding, and hormone binding. The DNA binding domain, which is required in order to activate transcription, consists of 66-68 amino acids of which about 20 sites, including nine cysteines (C1 to C9), are invariant among different receptors. The modular structure of members of this receptor superfamily allows the exchange of one domain for another to create functional, chimeric receptors.
The hormone response elements identified thus far are generally structurally related, but they are in fact functionally distinct. The response elements for GR [i.e., the glucocorticoid response element (GRE)], estrogen receptor [i.e., the estrogen response element (ERE)], and thyroid hormone receptor [i.e., the thyroid hormone response elements (TREs)] have been characterized in detail; they each consist of a palindromic pair of xe2x80x98half sitesxe2x80x99 [Evans, Science 240, 889 (1988); Green and Chambon, Trends in Genetics 4, 309 (1988)]. With optimized pseudo- or consensus response elements, only two nucleotides per half site are different in GRE and ERE [Klock, et al., Nature 329, 734 (1987)]. On the other hand, identical half sites can be seen in ERE and TRE, but their spacing is different [Glass, et al., Cell 54, 313 (1988)]. Moreover, TRE has been shown to mediate transcriptional activation by transfected retinoic acid receptors (RARs) in CV-1 cells whereas non-transfected cells show no response [Umesono et al., Nature 336, 262 (1988)]. In other words, both TR and RAR receptors can activate TREs.
Thus far, however, the response elements for only a few members of the steroid/thyroid superfamily of receptors have been identified. The response elements for many other members of the superfamily, and the relationship between them, if any, remain to be described.
We have discovered, and characterized by sequence, DNA segments which are response elements operative to confer responsiveness to ligands for several members of the steroid/thyroid superfamily of receptors, for the transcriptional activation and/or repression of promoters in cells. We have also discovered that the transcriptional activity modulating effect of the invention response elements occurs in all mammalian cells in the presence of ligands for several members of the steroid/thyroid superfamily of receptors, indicating that the various hormone receptors recognized by the invention response elements are present endogenously in all of these cells.
Contrary to what has previously been reported in the art for the GRE, ERE and TRE, the novel response elements disclosed herein have a tandem repeat sequence, as opposed to a palindromic sequence which has previously been reported for GRE, ERE and TRE. In addition, the invention response elements are much less susceptible to transcriptional activation by non-cognate receptors than are the previously described response elements (GRE, ERE, TRE).
By using transcriptional control regions comprising response elements of the present invention and a functional promoter, it is now possible to provide recombinant DNA vectors containing a gene, the transcription (and, thereby, also expression) of which is under the control of a promoter, the transcriptional activity of which is responsive to (and modulated by) ligands for several members of the steroid/thyroid superfamily of receptors.